Neuroprotective Potential of Sodium Orthovanadate in a Mouse Model of Chronic Unpredictable Mild Stress.

Depression is a psychiatric disorder characterized by low-esteem, anhedonia, social decit, and lack of interest. Decreased BDNF and impaired TrKB signaling have been found to be associated in depression. Moreover, oxidative stress in the brain and subsequent reduction of BDNF have a prominent role in psychiatric disorders. In our study, depressive-like behavior was induced in mice by chronic unpredictable mild stress (CUMS) model. Further, sodium orthovanadate (SOV), a protein tyrosine phospahatase was used as a test drug as it is reported to stimulate BDNF levels. Sodium orthovanadate (SOV-5 mg/kg, 10 mg/kg) and uoxetine (10 mg/kg) were given to mice orally for 21 days prior 30 minutes of stress induction. Various behavioral studies like tail suspension test (TST), open eld test (OFT), and sucrose preference test (SPT); biochemical analyses for corticosterone, reduced glutathione (GSH), lipid peroxidation (LPO), superoxide dismutase (SOD), nitric oxide (NO) and ELISA for BDNF were performed. Body weight was measured on a weekly basis. The behavioral tests reected depressive-like behavior in CUMS, which was attenuated by SOV and uoxetine. SOV (10 mg/kg) signicantly decreased malondialdehyde levels, NO, whereas increased GSH and SOD in both the cortex and hippocampus. Besides, ELISA revealed the elevation of BDNF levels in the treatment groups compared with the disease group (CUMS). Therefore, the treatment with SOV appeared to reverse both oxidative and nitrosative stress. Elevated BDNF level was associated with attenuation of depressive-like behaviors and serum corticosterone levels. The ndings of this preliminary study indicates that SOV has potential to restore mood and social interaction in depression and so further molecular mechanisms will be warranted for clinical translation. as compared to uoxetine group (Two-Way ANOVA followed by Bonferroni's post hoc test). CUMS: Chronic Unpredictable Mild Stress, SOV: Sodium Orthovanadate.


Introduction
Major Depressive Disorder (MDD) is one of the most prevalent, recurrent, and debilitating psychopathology forms. Epidemiological surveys indicate that the lifetime prevalence of MDD is 16.6%, with estimates as high as 21.3% in women (LeMoult and Gotlib 2019).
The monoamine hypothesis formulated in the 1990s suggested de ciency or imbalances in the monoamine neurotransmitters, such as serotonin (5-HT), dopamine (DA), and norepinephrine (NE), as the cause of depression. The antidepressants are prescribed to treat mild to severe depression. However, despite the increased synaptic content of monoamine neurotransmitters, tricyclic antidepressants and selective serotonin reuptake inhibitors produce their effect after a lag period. Moreover, they are useful in only 50% of patients.
Approximately 30-50% of patients don't even respond to their initial antidepressant trial, and the remission rates are as low as 37.5% (Bousman, Arandjelovic et al. 2019). This phenomenon probably results from the complex and multifactorial MDD etiology, which comprises psychosocial, biological, environmental, and genetic factors, explaining why most patients fail to respond to the standard monoaminergic antidepressants (de Sousa, V Zanetti et al. 2015). This limitation led to a paradigm shift towards the neurotrophin hypothesis, as depression is associated with neuronal atrophy and neuronal loss in speci c brain regions in several clinical and preclinical studies (Duman, Malberg et al. 2000, Mehrpouya, Nahavandi et al. 2015. Considering neurotrophins, the brain-derived neurotrophic factor (BDNF) is the major neurotrophin present in the central nervous system, which regulates neurogenesis (Numakawa, Odaka et al. 2017, Bai, Zhu et al. 2012 as it has a prominent role in the growth, differentiation, maturation, and survival of neurons. It also promotes the formation of dendritic spines and thus improves transmision e ciency of synapses by increasing their number. Hence it is vital for synaptic plasticity and augmentation of neurotransmission (Bai, Zhu et al. 2012). BDNF triggers the intracellular downstream signaling via multiple pathways i.e., phosphatidylinositol 3-kinase (PI3K), phospholipase Cyclase (PLC-), and mitogen-activated protein kinase (MAPK) (Sonoyama et al., 2020, (Bai, Zhang et al. 2019), (Minichiello, Calella et al. 2002). This results in the activation of cAMP response element-binding protein (CREB), modulating the expression of BDNF levels (Feng, Wang et al. 2019), thus improving synaptic plasticity and cell survival.
A number of clinical and preclinical studies have implicated the close association between BDNF and the mood disorders. It has been implicated that in depression, the BDNF-TrkB pathway gets impaired, resulting in the reduced secretion of BDNF (Nagahara and Tuszynski 2011, Marshall, Zhou et al. 2018), and the studies re ect that the treatment with antidepressant consequently increases the BDNF levels (Wolkowitz, Wolf et al. 2011). Therefore, BDNF has a stimulatory action on neurons by its interaction with high-a nity TrkB receptors at tyrosine residues. Stressful life events trigger HPA axis hyperactivation in about 70% of depressive patients (Yang, Zhao et al. 2015). Increased corticosterone levels associated with chronic stress (McGill, Bundle et al. 2006) impairs hippocampal BDNF function, which supports the hippocampal atrophy reported in major depression (Jacobsen and Mørk 2006). Stress also promulgate the production of proin ammatory cytokines in the brain microglia, resulting in reduced hippocampal neurogenesis (Belleau, Treadway et al. 2019 Notably, sodium orthovanadate (SOV) is an inorganic compound belonging to the vanadium family, having a role as protein tyrosine phosphatase inhibitor (PTP) (Cherry, Calder et al. 2017). Protein tyrosine phosphatase inhibition by SOV results in the activation of the PI3K/AKT and MAPK pathway, which further stimulates BDNF levels (Wang, Lu et al. 2015), improves cell survival, synaptic plasticity, and delay neuronal damage (Kawano, Fukunaga et al. 2001). Additionally, a study done on subarachnoid hemorrhage in rats has revealed that the administration of SOV resulted in the elevation of the BDNF levels, and SOV protected cortical and hippocampal neurons after experimental subarachnoid hemorrhage by increasing BDNF (Hasegawa, Suzuki et al. 2011). Also, sodium orthovanadate elicits an antioxidant property by regulating levels of SOD, GPx, catalase, LPO, and glutathione in the diabetic rat (Sekar, Kanthasamy et al. 1990).
With the above background, depicting the role of BDNF-Trk signaling in major depressive disorder and the potential of SOV in improving BDNF signaling, we hypothesized that SOV can have a bene cial effect which was evaluated in CUMS induced rodent model of depression.

Experimental animals
Male Balb/c male mice with a weight ranging between 29-34 g were procured from Central Research Institute Kasauli and Central Animal House of Panjab University Chandigarh, India. All the mice were housed in a room at temperature 25 ± 2°C with proper light-dark periods with water and food provided ad libitum. Whole interventional protocols were conducted between the period from 09:00 to 17:00 according to the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The study protocol was approved by the Institutional Animal Ethical Committee (IAEC) of Panjab University, Chandigarh, with an approval number of PU/45/99/CPCSEA/IAEC/2019/303.

Drugs and treatments
Sodium orthovanadate (SOV) (catalog no.: S6508-10G) and uoxetine (FLX) were procured from Sigma-Aldrich (St. Louis, MO, United States). Distilled water was used to dissolve SOV and administered orally with the help of an oral gavage. Normal saline, i.e., 0.9% NaCl, was used to dissolve the uoxetine and then given orally in a dose of 10 mg/kg (Machado, Cunha et al. 2012). Distilled water was used as the solvent in the preparation of SOV and administered orally. Doses were decided in accordance with the previously reported studies (Hasegawa, Suzuki et al. 2011,Akhtar, Bishnoi et al. 2020) which have shown an attenuation of neuronal death and oxidative damage along with alleviation of mitochondrial dysfunction. Mouse ELISA kit for BDNF estimation was procured from Elabscience (catalog no.: E-EL-M0203), USA. The whole treatment was given daily starting from day 8th to 28th day.

Experimental design
Animals (n = 42) were randomly divided into seven groups, containing an equal number of animals (n = 6) as dipicted in Table 1.
Initially, for 1st week, normal saline water (0.9% NaCl) was given to the control group (that was not subjected to CUMS) and the CUMS group. From day 8th, treatment was given to all the groups prior 30 minutes of stress induction (CUMS group and per-se group) till 28th day. On 29th and 30th day sucrose preference test (SPT) was performed, whereas a tail suspension test (TST) was performed on the 31st day. To check the effect on locomotor activity, open eld test (OFT) was performed on 32nd day. The duration of the protocol lasted for 33 days from the induction of CUMS model until the sacri ce of animals and body weight was analyzed weekly. On the 33rd day, blood was collected from the animals under anesthesia, and then animals were euthanized for isolation of cortex and hippocampus to perform biochemical analysis and ELISA (Fig. 1). Animal stress models are commonly used in the preclinical antidepressant evaluation. Chronic unpredictable mild stress (CUMS) has been widely used in animals to mimic depressive-like behavior and is regarded as being close to the unexpected stressors of everyday life in humans. Animals were exposed to CUMS for 28 days except control and per-se group. The different types of stressors were given to the animal both on a regular and repetition basis till the sacri ce of animals as depicted in the  To nd out any effect of the drug on locomotor activity, OFT was conducted. The number of lines crossed was evaluated in the open eld paradigm under normal daylight. The whole procedure was performed as per the previously described study (Idayu, Hidayat et al. 2011). Before the commencement of OFT, mice were acclimatized to environment for 2-3 min. The test apparatus consisted of an arena with a measurement of 50 cm x 30 cm and painted with black colour. The oor of the test apparatus consisted of 25 virtually produced grids made with the help of Ethovision. Each mouse was placed in the middle of the arena and then allowed to explore freely. The number of lines crossed by the mice with all their paws within each grid in 6 min was evaluated. After each testing, the apparatus was cleaned with 70% ethanol to remove any odor and clues of previous mouse made by its urine and fecal content. Results were expressed as number of lines crossed.

Sucrose preference test (SPT)
A state of anhedonia characterizes depression, and lowered sucrose consumption in rodents is a clear-cut indication of this state. The procedure was performed as described previously (Wang, Cui et al. 2011). Brie y, 30 h before the test, mice were deprived of water and food, then 2 bottles were placed in the cage containing 1% sucrose solution (w/v) and regular tap water, respectively. Animals were freely allowed to access both the bottles for 24 h. At the end of 24 h, the sucrose preference (%) was calculated as below. Results were expressed as the percentage of sucrose consumed.

Tail suspension test (TST)
This test was performed to assess learned helplessness in mice as per the previously described procedure (Kulkarni and Dhir 2007). Prior to conduction of TST, all mice were acclimatized to surroundings for 2-3 min. Mice were isolated from any external sound and visuals and placed 50-55 cm above the ground by xing the tail on the frontal lever (approx. 1 cm from tip) with adhesive tape. During the total 6 min of test, acclimatization was done for initial 2 min, and the remaining time was utilized for recording the immobility of mice on kymograph. Results were expressed as immobility time (sec).

Body weight measurement
Animal body weight was measured every week of the experimental protocol. Fluctuation in body weight was calculated based on data acquired weekly. Results were expressed as grams.

Tissue homogenate preparation
After completing all the behavioral evaluations, the animals were sacri ced under anesthesia with ketamine (70 mg/kg) and xylazine (10 mg/kg) combination. During anesthesia, the blood was collected through the retroorbital plexus and stored in the eppendorf containing EDTA. Afterward, the animals were sacri ced; brains were isolated and perfused with PBS. Cortex and hippocampus were isolated later from the whole brain. The isolated tissues were stored and homogenized in 10% (w/v) homogenization buffer (comprising of 10 mM Tris-HCl, 150 mM MgCl 2 , 1mM EDTA, 1% Triton X 100, pH equivalent to 7.4) and centrifuged at 10,000 rpm and 4º C for 20 min. After the centrifugation, the supernatant was isolated by pipette and stored at − 80º C for various antioxidant assays and ELISA. Further, the plasma was separated by the process of centrifugation at 10,000 rpm for 10 min and stored at -80ºC for further estimations.

Estimation of Protein
The biuret method was used for the quanti cation of protein (Gornall, Bardawill et al. 1949). The standard curve of bovine serum albumin was used to determine protein concentration expressed in mg/ml. Thus, the values obtained were used in the calculations of other biochemical results.

Reduced glutathione (GSH) assay
Reduced glutathione was estimated based on a previous study (Jollow, Mitchell et al. 1974). In the method, 100 µl of the supernatant of tissue homogenate was added to 1 ml of 4% w/v sulfosalicylic acid. The precipitate was formed, and the reaction mixture was kept at temperature 2-8°C in the refrigerator. After one hour, samples were centrifuged in a cold centrifuge, i.e., 4°C, at a rotation of 1200 g for 15 min. Pellet was discarded to obtain 2.6.5 Estimation of superoxide dismutase (SOD) SOD was estimated as per described study (Kono 1978

Estimation of plasma corticosterone
The plasma corticosterone estimation was done as previously described (Bartos and Pesez 1979). For the assessment of corticosterone levels in the blood (plasma), the reagents respectively reagent A (0.

Statistical Analysis
Analysis of data was done using a one-way ANOVA or two-way ANOVA followed by a Tukey's post hoc test or Bonferroni's post hoc test respectively for multiple comparisons. Statistically signi cant effects were de ned as those with levels of P-values < 0.05. The standard error of the mean was represented by error bars. Prism CUMS for 28 consecutive days signi cantly (P < 0.001) decreased the ambulatory score as compared to the control group. Treatment with SOV (10 mg/kg) and uoxetine signi cantly increased the ambulatory score when compared to CUMS group (P<0.05). Treatment with a combination group showed no signi cant difference in the ambulatory score than the CUMS and control group. The per-se group did not show any difference compared with the control group (Fig. 2

Effect of SOV, uoxetine and their combination on the level of GSH, LPO, and activity of SOD
In both the hippocampus and cortex areas in CUMS group, the levels of GSH were decreased (P<0.001) signi cantly compared to the control group. Treatment with sodium orthovanadate (5 mg/kg and 10 mg/kg) signi cantly increased the levels of GSH in both cortex (P<0.05 and P< 0.01) and hippocampus (P< 0.05).
Fluoxetine showed a signi cant increase in the levels of GSH in both cortex and hippocampus compared to the CUMS group (P<0.05). Per-se group did not show any signi cant result in the GSH levels compared to the control group, both in the hippocampus and cortex (Fig. 5).
The level of LPO in CUMS group was found to be signi cantly increased in both cortex (P < 0.001) and hippocampus (P< 0.05) as compared to the control group. Treatment with sodium orthovanadate (10 mg/kg), uoxetine, and combination group signi cantly decreased the levels of LPO as compared to CUMS group in both cortex (P< 0.01) and hippocampus (P< 0.05). In addition to the above, sodium orthovanadate (5 mg/kg) signi cantly decreased the levels of LPO in the cortex region (P< 0.001). In both cortex and hippocampus, the lipid peroxidation did not increase in the per-se group compared to the control group (Fig. 6).
The antioxidant enzyme SOD was signi cantly decreased in both the hippocampal and cortical regions of the brain in CUMS group compared to control mice (P < 0.001). Treatment with sodium orthovanadate (10 mg/kg) showed a signi cant increase in the level of SOD in both hippocampus (P<0.01) and cortex (P< 0.05) as compared with CUMS group. Fluoxetine showed a signi cant increase in the SOD levels in the hippocampus as compared to CUMS group (P< 0.001), and also SOD levels in the combination group were found to be increased signi cantly in the hippocampus as compared to CUMS group (P<0.01). Per-se group signi cantly decreased the SOD levels in both hippocampus and cortex as compared to the control group (P< 0.05) (Fig. 7).

Effect of SOV, uoxetine and their combination on the levels of plasma corticosterone and NO
The plasma corticosterone levels were signi cantly increased in CUMS group compared to the control group (P < 0.001). Treatment with sodium orthovanadate (5mg/kg and 10 mg/kg), uoxetine, and combination group signi cantly decreased the levels of plasma corticosterone as compared to CUMS group (P< 0.001). The per-se group produced a signi cant decrease in plasma corticosterone levels as compared to the control group (P< 0.001) (Fig. 8). However, SOV (10 mg/kg) in both cortex (P< 0.05) and hippocampus (P< 0.001) area signi cantly decreased NO levels as compared to CUMS group. Per-se group signi cantly increased the levels of nitrite as compared to the control group (P< 0.001) (Fig. 9).

Effect of SOV and uoxetine on the levels of brain-derived neurotrophic factor
Levels of BDNF were decreased signi cantly in both cortex and hippocampus compared to the control group (P< 0.01). Treatment with SOV (5 mg/kg and 10 mg/kg) and uoxetine signi cantly increased the BDNF levels in both hippocampus and cortex as compared to CUMS group (P< 0.05) (Fig. 10).

Discussion
In our present study, we used sodium orthovanadate (SOV) to establish the role of BDNF and oxidative stress in depression. Moreover there is an inverse relationship between them, re ecting a possible mechanism of interaction between oxidative stress and neurotrophin dysfunction (Zhang, Chen et al. 2015). Corticosterone, a stress hormone, is an indicator of the anxiety and depressive-like behavior in an individual. Stress elevates corticosterone levels by activating the HPA axis, resulting in neuronal atrophy arising due to decreased brain levels of neurotrophins like BDNF. Corticosterone decreases BDNF mRNA expression gradually, resulting in diminished levels of BDNF protein translation (Liu, Walther et al. 2005). BDNF is one of the growth factors that trigger neuronal survival after BDNF-TrkB signaling. Impairment in the neurotrophins, mainly BDNF, leads to depressive-like behavior, increased hippocampal dendritic atrophy, cell death, and reduced LTP. Impaired BDNF expression has also been reported in MDD. A study has revealed that cortisol levels get elevated, and serotonin levels get declined on the induction of chronic stress via serotonin reuptake, further resulting in the depressive- neurogenesis. Therefore, considering this CUMS paradigm, which is a well-validated model of depression produced by the set of stressors in rodents (Schildkraut 1965), the present study was used to elucidate the impaired role of BDNF levels in CUMS induced depressive like behavior and its amelioration with SOV.
CUMS signi cantly downregulates the levels of BDNF and CREB, resulting in a depressive-like behavior (Levi-Montalcini 1950). Increased corticosterone levels and decreased levels of BDNF after CUMS in our results are in line with the above ndings. However, our study also revealed that the treatment with SOV signi cantly decreased the plasma corticosterone levels. The effect produced by SOV per-se on corticosterone levels is consistent with previous ndings where vanadium compound attenuated corticosterone levels in rats (Katayama, Yamada et al. 2010). According to a prior study, the chronic FLX treatment in CUMS exposed rats also normalized the corticosterone levels ( CUMS model shows a declined sensitivity to reward, termed as the anhedonic state (Hutchinson, Chou et al. 2009), one of the core symptoms of depression. A study con rmed that mice exposed to CUMS consume less sucrose uid (Fukuchi 2020), supporting our ndings. An open eld test was conducted to analyze the locomotor activity and explore the novel environment (Wills 1965). Many studies depict the decline in the number of line crossing activity (OFT) in CUMS group, re ecting a depressive-like behaviors (Fukuchi 2020).
Our study also showed a similar nding re ecting the effect of CUMS in the induction of a depressive-like behaviors. However, both the SOV doses in the present study improved the core features of depression in rodents like anhedonia, despair behavior, and hypo-locomotion. SOV per-se resulted in decreased sucrose consumption that could result from its roles in improving leptin and insulin signaling, which play an essential role in regulating energy balance through food-associated reward control (Zhang, Chen et al. 2015 (Mehrpouya, Nahavandi et al. 2015). In the present study, we analyzed MDA levels (an indicator of lipid peroxidation), NO, GSH, and SOD in the hippocampal and cortical tissues of mice exposed to CUMS. CUMS exposure resulted in the generation of oxidative stress and nitrosative stress in both the cortex and hippocampus. However, chronic SOV treatment ameliorated oxidative and nitrosative stress in the brain, emphasizing the role of BDNF in mediating antioxidant effects (Bibring 1953  Neuronal degeneration is a signi cant consequence of ROS generation. In vitro studies suggested that ROS act in a neurotoxic as well as in a neuroprotective manner, which is enhanced by TrkB (Harlow, Newcomb et al. 1986). However, ROS has a signi cant role in psychiatric disease due to the vulnerability of the central nervous system to oxidative stress and CUMS results in induction of ROS expression by Akt pathway modulation (Mulinari 2012). In addition to the above nding, a study conducted on cisplatin-induced ROS suggested that BDNF attenuates ROS generation, resulting in the decline in ROS levels (Carroll 1971). Indeed, a speci c pathway has not been evolved fully to support the link between ROS and TrkB.
Meanwhile, a study conducted on cell lines revealed that vanadate compound per-se could generate ROS resulting in the decreased levels of SOD via MAPK pathway activation (Fawcett and Barkin 1997). And our study also implies that SOV per-se decreased SOD and increased nitrite levels per-se while it does not affect MDA levels. Whereas it produced a signi cant antioxidant effect in CUMS exposed rats supported by previously conducted study (Kim, Lee et al. 2019).
Our study revealed that CUMS exposure increased nitrite levels, whereas SOV (per-se) also increased nitrite levels via the Akt pathway's activation (Aid, Kazantseva et al. 2007) while SOV at a dose of 10 mg/kg signi cantly decreased its level. CUMS activates microglia, which gets activated to produce in ammatory cytokines. These in ammatory markers are the leading cause for the production of nitrites in the brain whereas protein tyrosine phosphatase 1B (PTP1B), a member of the protein tyrosine phosphatases (PTPs) family, positively regulates neuroin ammation by causing dephosphorylation of proteins at tyrosine residues. SOV, a PTP inhibitor reduces this feature induced by CUMS, resulting in a decline in nitrite levels than the CUMS group (Seifer, Feng et al. 2006